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Three generations of DNA testing. RFLP AUTORAD Allele = BAND. DQ-alpha TEST STRIP Allele = BLUE DOT. Automated STR ELECTROPHEROGRAM Allele = PEAK. How do they fare?. STR. DQ-alpha. RFLP.  Discriminating power  Sensitivity □ Technical artifacts  Speed □ Mixtures

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Three generations of dna testing
Three generations of DNA testing

RFLP

AUTORAD

Allele = BAND

DQ-alpha

TEST STRIP

Allele = BLUE DOT

AutomatedSTR

ELECTROPHEROGRAM

Allele = PEAK


How do they fare
How do they fare?

STR

DQ-alpha

RFLP

 Discriminating power

Sensitivity

□ Technical artifacts

 Speed

□ Mixtures

 Databasing

 Discriminating power

□ Sensitivity

□ Technical artifacts

□ Speed

□ Mixtures

ٱDatabasing

□ Discriminating power

 Sensitivity

Technical artifacts

 Speed

□ Mixtures

□ Databasing


Three generations of dna testing

RFLP technology

What we would

like for them to look like.


Three generations of dna testing

What they often

look like.

Incomplete digest


Available kits for str analysis
Available Kits for STR Analysis

  • Kits make it easy for labs to just add DNA samples to a pre-made mix

  • 13 CODIS core loci

    • Profiler Plus and COfiler (PE Applied Biosystems)

    • PowerPlex 1.1 and 2.1 (Promega Corporation)

  • Increased power of discrimination

    • CTT (1994): 1 in 410

    • SGM Plus™ (1999): 1 in 3 trillion

    • PowerPlex ™ 16 (2000): 1 in 2 x 1017


Overview of steps involved in dna typing

TPOX

TH01

CSF

D7

D16

D3

D5

D13

Penta D

D8

D18

FGA

VWA

D21

AMEL

Penta E

Blood Stain

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

DNA Quantitation using Slot Blot

Genotyping by Comparison to Allelic Ladder

Overview of Steps Involved in DNA Typing


Repeated dna
REPEATED DNA

  • Satellite DNA

    • Around chromosomal centromere

    • Long repeats can be 100s to 1000s bp long

  • Minisatellite DNA

    • VNTR

    • Medium repeats 10 to 100 bp long

  • Microsatellites

    • STR

    • Short repeats 2 to 6 bp long


Short tandem repeats
SHORT TANDEM REPEATS

  • Easy to amplify

  • Both heterozygote alleles amplify well

  • Number of repeats highly variable

  • Good for identification

  • Many different sites


Str nomenclature
STR NOMENCLATURE

  • Simple Repeats

    • Identical length and sequence

      • agat agat agat agat agat

  • Compound Repeats

    • Two or more adjacent simple repeats

      • agat agat agat ttaa ttaa ttaa

  • Complex Repeats

    • Variable unit length & possible intervening seq

      • agat agat aggat agat agat ttaacggccat agat agat


The 13 codis str loci
THE 13 CODIS STR LOCI

  • Four categories

  • Simple repeats

    • TPOX, CSF1PO, D5S818, D13S317, D16S539

  • Simple repeats w/ non-consensus alleles

    • TH01, D18S51, D7S820

  • Compound repeats w/ non-consensus alleles

    • vWA, FGA, D3S1358, D8S1179

  • Complex repeats

    • D21S11


Commercial str kits
Commercial STR Kits

  • Applied Biosystems

  • AmpFlSTR Profiler Plus

    • 5-FAM (BLUE)

    • Joe (GREEN)

    • NED (YELLOW)

  • AmpFlSTR Cofiler

    • 5-FAM (BLUE)

    • Joe (GREEN)

    • NED (YELLOW)

D3S1358

vWA

FGA

Amelo

D8S1179

D21S11

D18S51

D5S818

D13S317

D7S820

D16S539

D3S1358

Amelo

TH01

TPOX

CSF1PO

D7S820


Commercial str kits1
Commercial STR Kits

  • Promega Corporation

  • PowerPlex 1.1

    • Fluorescein (Blue)

    • TMR (Yellow)

  • PowerPlex 2.1

    • Fluorescein (Blue)

    • TMR (Yellow)

D5S818

D13S317

D7S820

D16S539

vWA

TH01

TPOX

CSF1PO

D3S1358

TH01

D21S11

D18S51

Penta E

Amelo

vWA

D8S1179

TPOX

FGA



Pcr amplification with fluorescent str kits and separation with capillary electrophoresis

TPOX

TH01

CSF

D7

D16

D3

D5

D13

Penta D

D8

D18

FGA

VWA

D21

AMEL

Penta E

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis



An example forensic str multiplex kit

AmpFlSTR® Profiler Plus™

Kit available from PE Biosystems (Foster City, CA)

200 bp

300 bp

400 bp

100 bp

Size Separation

D3

vWA

FGA

5-FAM (blue)

JOE (green)

A

D8

D21

D18

Color Separation

D5

D13

D7

NED (yellow)

ROX (red)

GS500-internal lane standard

9 STRs amplified along with sex-typing marker amelogenin in a single PCR

An Example Forensic STR Multiplex Kit


Three generations of dna testing

Human Identity Testing with Multiplex STRs

AmpFlSTR® SGM Plus™ kit

DNA Size (base pairs)

D3

TH01

amelogenin

D8

VWA

D16

D19

D21

D18

D2

FGA

probability of a random match: ~1 in 3 trillion

Two different individuals

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

D3

amelogenin

D19

D8

D16

VWA

D21

D18

D2

FGA

TH01

Simultaneous Analysis of 10 STRs and Gender


Fbi s codis dna database
FBI’s CODIS DNA Database

Combined DNA Index System

  • Launched October 1998

  • Used for linking serial crimes and unsolved cases with repeat offenders

  • Links all 50 states

  • Requires >4 RFLP markers and/or 13 core STR markers

  • Current backlog of > 600,000 samples

    As of June, 2004

  • Total profiles = 1,857,093

  • Total forensic profiles = 85,477

  • Total convicted offender = 1,771,616


Population database
Population database

  • Look up how often each allele occurs at the locus in a population (the “allele” frequency)